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Effect of the P838L mutation on myosin function in Drosophila melanogaster
Bernstein, Sanford I.
Harris, Greg L.
Myosin, a motor protein, is composed of two heavy chains and four light chains. It hydrolyzes ATP to generate actin-based motility and the contractile force in muscles. A mutation in human cardiac beta myosin heavy chain that changes amino acid residue 838 from proline to leucine (P838L) results in pediatric restrictive cardiomyopathy. Restrictive cardiomyopathy (RCM) leads to rigidity of the ventricular wall and the heart is restricted from stretching and properly filling with blood. Therefore, blood flow is reduced and blood that would normally enter the heart is backed up in the circulatory system. Gradually, the patient loses the ability to pump blood efficiently, leading to heart failure. Drosophila has a rhythmically beating heart and serves as a powerful tool to study the genetic basis of heart development and disease in humans. To define the biochemical basis of myosin-based RCM and to test the hypothesis that the P838L mutation causes RCM in Drosophila, a gene encoding myosin with the P838L mutation was constructed and was expressed in place of wild-type myosin heavy chain by P element transformation. Jump tests indicated that the mutation did not have any effect on the jump muscles. However reduced flight ability was observed, indicating impairment in the indirect flight muscle expressing this mutant myosin. ATPase assays and in vitro motility assays were performed to determine the effect of the mutation at the molecular level. We found that there was a slight increase in the actin-sliding velocity and that the basal and actin-activated MgATPase activity was significantly higher for the mutants. Electron microscopy on 2 day-old mutants and the controls suggested that the myofibrils were intact but some of them were oblong in shape and had rough edges when compared to the control myofibrils. Preliminary video microscopy data suggest that the transgenic hearts display a restrictive phenotype. The P838L mutation affects an "invariant proline" located at the junction of the myosin S1 head and the S2 rod and might affect the movement of the myosin heads during the force generation step. This could lead to the observed increase in the ATPase rate. Also, the P838L mutation is located near to where the regulatory light chain (RLC) binds to the heavy chain. RLC has an important role in the ATPase cycle and the P838L mutation might alter the RLC conformation leading to the increased ATPase rate. The increased ATPase rate seen in this and another Drosophila myosin that causes a restrictive phenotype may initiate a cascade of events that result in the observed cardiac defects.
Master of Science (M.S.) San Diego State University, 2012
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